Protective effects of SnF₂ – Part I. Mineral solubilisation studies on powdered apatite

Abstract

Purpose: To compare the ability of two active ingredients – sodium fluoride (NaF) and stannous fluoride (SnF₂) – to inhibit hydroxyapatite (HAP) dissolution in buffered acidic media. Methods: Two in vitro studies were conducted. HAP powder, which is representative of tooth mineral, was pretreated with: test solutions of NaF or SnF₂, 10 g solution per 300 mg HAP powder (Study 1); or NaF or SnF₂ dentifrice slurry supernatants, 20 g supernate per 200 mg HAP powder for 1 minute followed by three washes with water, then dried (Study 2). About 50 mg of pretreated HAP was exposed to 25 ml of acid dissolution media adjusted to and maintained at pH 4.5 in a Metrohn Titrino reaction cell. Exposure of HAP to the media results in dissolution and release of hydroxide ion, increasing the pH of the solution. The increase in pH is compensated for by automatic additions of acid to maintain the original pH (4.5) of the reaction cell. Total volume of titrant added after 30 minutes was used to calculate the percentage reduction in dissolution versus non-treated HAP control. Results: Both F sources provided protection against acid dissolution; however, in each study, SnF₂-treated HAP was significantly more acid-resistant than the NaF treated mineral. In study 1, at 280 ppm F, representing concentrations of F found in the mouth after in vivo dentifrice use, the reduction in HAP dissolution was 47.7% for NaF and 75.7% for the SnF₂-treated apatite (extrapolated). In study 2, the reduction in HAP dissolution was 61.3% for NaF and 92.8% for SnF₂-treated samples. Differences in percentage reduction were statistically significant (Paired-t test). Conclusions: Results of these studies demonstrate that both of the fluoride sources tested enhance the acid resistance of tooth mineral and that resistance is significantly greater after treatment with SnF₂ compared with treatment of tooth mineral with NaF.

INTRODUCTION

Protection against erosive acid challenge is critical for the prevention of dental erosion. Stannous fluoride (SnF₂), a well-known anticaries agent, has also been shown to impart significant resistance to tooth mineral against erosive acid attack1., 2., 3., 4.. This effect is believed to be driven by the strong affinity of stannous ions for the mineral surface on teeth¹. In vitro studies, using hydroxyapatite (HAP) powder, were used to demonstrate the mechanism of SnF₂ protection against dental erosion. Hydroxyapatite, the primary mineral component of human enamel, provides an efficient means to determine the ability of compounds to attach to and protect tooth mineral against erosive acid dissolution⁵.

It has been over 60 years since SnF₂ was first suggested as an agent for reducing the acid solubility of tooth enamel as a mechanism for the prevention of dental caries6., 7.. Since that time, the effectiveness of SnF₂ in lowering the incidence of caries has been shown in numerous clinical trials⁸. The ability of the stannous ion to deliver enamel solubility reduction was noted in several publications in the early days of research on SnF₂6., 9., with one perspective being that the clinical effectiveness of the agent was likely dependent on the maintenance of the stannous ion in an active state¹⁰. Electron microscopic studies demonstrated that stannous ions could be deposited on intact enamel surfaces¹¹. Other in vitro studies demonstrated the separate but additive protective effects of the stannous and fluoride ions against acid solubility of enamel¹². However, the specific mechanism by which this protection was delivered was not clearly established¹³. Brudevold and co-workers9., 14. found little or no stannous ion uptake on the surface of teeth exposed to a SnF₂ dentifrice, while Wachtel¹² reported no evidence of significant stannous ion protection using in vitro enamel solubility tests. Meanwhile, Muhler¹⁵ demonstrated that daily application of SnF₂ dentifrice increased the tin content of enamel, and Manly¹⁶ showed that the enamel solubility rate was related to the availability of stannous ion. Despite the differences in opinion regarding a specific mechanism of action, there was little doubt SnF₂ was clinically effective in the control of caries. Importantly, the preponderance of evidence suggested that stability of the stannous ion played a major role in reducing the solubility of tooth enamel in acid.

Over the past several decades, new information has come to light regarding both the chemistry of SnF₂ and methods for stabilisation of this highly reactive species into oral care products. Stannous fluoride is unstable in aqueous media and above pH 4 undergoes oxidation and hydrolysis reactions resulting in the formation of inactive stannous species and precipitates, leading to significant reductions in caries, gingivitis and dentinal hypersensitivity benefits. To compensate for the loss of stannous ion, dentifrice manufacturers stabilise SnF₂ through the use of various formulation modifications, such as exclusion of water, use of chelating agents, such as pyrophosphate, gluconate, gantrez (a copolymer of maleic acid and methyl ether) or phytate, which are able to form soluble stannous complexes, or through the incorporation of another stannous compound, such as stannous chloride or stannous pyrophosphate as a reserve source of stannous ions¹⁷. Through careful formulation balancing and performance modelling, manufacturers have developed the ability to deliver products that now provide high levels of both stannous and fluoride ions during product use¹⁸.

Owing to their ability to provide high levels of available active, stabilised SnF₂ dentifrices have gained significant respect in the dental community for their ability to not only fight caries but also to control plaque¹⁹, gingivitis²⁰ and dentinal hypersensitivity21., 22., 23.. More recently, both in vitro1., 2. and in situ clinical studies3., 4. have demonstrated the superior ability of a stabilised SnF₂ dentifrice to also protect teeth against erosive acid attack that can lead to dental erosion. Dental erosion is a condition arising from an overexposure of teeth to dietary acids that can result in tooth surface damage. Whereas caries results in loss of subsurface tooth mineral, dental erosion attacks the surface of teeth and can result in irreversible tooth surface loss²⁴. The ability of SnF₂ to protect tooth mineral against erosive acid challenges begs the question of whether its anti-erosion mechanism of action is similar to or different from its anti-caries mechanism of action.

The purpose of the current studies was to measure, in the absence of dentifrice components such as emulsifiers, humectants, detergents and flavours, the relative ability of freshly prepared NaF and SnF₂ solutions to reduce acid solubilisation of powdered HAP (Study 1) and whether the relative ability of these active ingredients to reduce acid dissolution changes when incorporated into modern dentifrice formulations (Study 2). An in vitro HAP dissolution model was used to measure in vitro dissolution kinetics of pretreated HAP, a synthetic mineral analogue of teeth, under acidic solution conditions.

METHODS

The model uses HAP powder, a synthetic analogue of enamel and dentine, as a test substrate. The HAP powder (BioRad Laboratories, Hercules, CA, USA) was pretreated with a freshly made solution [Study 1: 10 g of NaF or SnF₂ solutions (over the range of 12.5–400 ppm F) per 300 mg HAP powder] or dentifrice slurry supernatant (Study 2: 20 g of supernate per 200 mg HAP powder) followed by washing three times with water, then dried. About 50 mg of the pretreated HAP powder was then exposed to 25 ml of acid dissolution media. The pH of the media was adjusted to and maintained at pH 4.5. Exposure of HAP to the media results in dissolution and release of hydroxide ions, which increases the pH of the solution:

$G_{\mathrm{mr}}=\frac{G_{\mathrm{pre}}+G_{\mathrm{post}}}{2}$

Compensation for increasing pH is made by sequential, automatic additions of 0.05 m lactic acid (Study 1) or dilute (0.1 n) hydrochloric acid (Study 2) to maintain the original pH (4.5) of the reaction cell (Metrohm Titrino – Figure 1; Brinkman Instruments, Riverview, FL, USA). As there is a finite amount of solution present, the rate of dissolution and subsequent amount of titrant added slows over time. The total volume of titrant added after 30 minutes was used to calculate per cent reductions versus control.

Figure 1.

Metrohn Titrino reaction cell system.

Preparation of treatment solutions (Study 1)

For the NaF treatment solutions a stock solution of 1000 ppm F (2.21 g NaF diluted to 1,000 ml) was prepared fresh in a Nalgene volumetric flask (Nalgene, Rochester, NY, USA) using reagent grade NaF powder and deionised, distilled water. Serial dilutions were made from this stock solution to concentrations of 25, 50, 100, 200 and 400 ppm F using deionised, distilled water.

For SnF₂ treatment solutions a stock solution of 1,000 ppm F (4.124 g SnF₂ diluted to 1000 ml) was prepared fresh in a Nalgene volumetric flask using reagent grade SnF₂ powder and deionised, distilled water. Serial dilutions were made from this stock solution to concentrations of 12, 24, 49, 73, 121 and 243 ppm F using deionised, distilled water.

About 300 mg of HAP powder was added to 10 g of each treatment solution and thoroughly mixed for 1 minute to allow reaction between the apatite surfaces and the treatment solutions. Treatment solutions were then filtered and washed a total of three times using deionised, distilled water. The filtrate was recovered, dried and stored until ready for use in the dissolution experiments.

About 50 mg of the pre-treated HAP powder was added to 25 ml of dissolution media (0.10 m lactic acid + 0.15 m NaCl, pH 4.5) in the reaction cell (Metrohm Titrino). Titrant for reaction cell: 0.05 m lactic acid.

Preparation of treatments (Study 2)

Dentifrice slurries were prepared using commercially available dentifrices. Crest Cavity Protection dentifrice (The Procter & Gamble Company, Cincinnati, OH, USA), containing 1,100 ppm F as NaF and Crest Pro-Health dentifrice (The Procter & Gamble Company), containing 1,100 ppm F as SnF₂ were used. Dentifrice slurries were prepared by thoroughly mixing one part dentifrice (10.0 g) with three parts (30.0 g) deionised, distilled water for approximately 2 minutes. After mixing, slurries were transferred to a 50 ml Nalgene tube and centrifuged at 12,400 g for 30 minutes to produce a supernatant. Twenty grams of the supernatant were removed for use in the study. Duplicate supernatants were prepared for each test product. About 200 mg of HAP powder was added to 20 g of each supernatant and thoroughly mixed for 1 minute to allow reaction between the apatite surfaces and the treatment. Treated HAP powders were then filtered, and washed a total of three times using deionised, distilled water. The recovered filtrate was freeze dried and stored until ready for use in the dissolution experiments.

About 50 mg of the pretreated HAP powder was added to 25 ml of dissolution media (0.05 m acetic acid, 0.05 m sodium acetate, 0.109 m sodium chloride) pH 4.5, in the reaction cell (Metrohm Titrino). Titrant for reaction cell: 0.10 n hydrochloric acid.

RESULTS

Both NaF and SnF₂ provided protection against acid dissolution, with results significantly favouring performance by the SnF₂ in each study. These results further demonstrated that the SnF₂ remains active in the modern, stabilised formulation, providing a significant improvement over earlier dentifrices that suffered from stability issues. In Study 1, freshly prepared SnF₂ and NaF solutions both reduced the dissolution rate of HAP powder compared with a non-treated HAP control. The percentage reduction in dissolution after 30 minutes (Table 1) was correlated with Log[concentration] of each ingredient (Figure 2). For the SnF₂ series, % Reduction Y = 15.867Ln[F conc.] − 13.703, R² = 0.9884; for the NaF series, % Reduction Y = 6.68Ln[F conc.] + 9.9915, R² = 0.9596. At a given F concentration, e.g. 280 ppm, which represents the expected concentration of F found in the mouth after in vivo dentifrice use, the reduction in HAP dissolution for NaF was 47.6%, while reduction for the SnF₂-treated sample was 75.7% (extrapolated – Table 2, Figure 2). In study 2, after 30 minutes of acid exposure (Table 3), the reduction in dissolution for the NaF-treated apatite was 61.3%, while the reduction in HAP dissolution for the stabilised SnF₂-treated sample was 92.8% (Table 4, Figure 3). Differences in % reduction were statistically significant (paired t-test).

Table 1.

pH stat data (Study 1) – (a) NaF solutions, (b) SnF₂ solutions

Time (minutes)	Parts per million (ppm) F in solution
	0	12.5	25	50	100	200	400
(a)
0.0	0	0	0	0	0	0	0
1.0	3.45	3.28	3.14	2.80	2.36	2.35	2.39
2.0	5.98	5.38	5.09	4.52	3.93	3.82	3.84
3.0	7.79	6.75	6.33	5.64	4.97	4.78	4.77
4.0	9.15	7.72	7.20	6.41	5.70	5.44	5.41
5.0	10.21	8.41	7.83	6.96	6.23	5.94	5.87
10.0	12.99	9.81	9.08	8.03	7.30	6.88	6.68
15.0	13.74	10.22	9.49	8.40	7.68	7.26	7.02
20.0	14.08	10.44	9.73	8.62	7.94	7.51	7.22
25.0	14.27	10.59	9.90	8.77	8.12	7.68	7.38
30.0	14.39	10.70	10.03	8.88	8.25	7.80	7.49

Time (minutes)		Parts per million (ppm) in solution
	Sn	0	38	76	151	227	379	757
	F	0	12	24	49	73	121	243
(b)
0.0		0	0	0	0	0	0	0
1.0		3.39	3.37	2.83	2.49	1.88	1.52	0.94
2.0		5.76	5.34	4.49	4.02	3.31	2.61	1.61
3.0		7.43	6.56	5.53	4.95	3.91	3.30	2.06
4.0		8.67	7.39	6.23	5.57	4.42	3.76	2.27
5.0		9.62	7.98	6.72	6.01	4.70	3.99	2.41
10.0		12.09	9.02	7.56	6.72	5.26	4.52	2.85
15.0		12.79	9.35	7.89	6.99	5.53	4.77	3.08
20.0		13.12	9.55	8.09	7.17	5.69	4.92	3.23
25.0		13.31	9.68	8.23	7.30	5.81	5.04	3.35
30.0		13.43	9.78	8.33	7.40	5.90	5.13	3.45

Figure 2.

Hydroxyapatite dissolution kinetics; comparison of aqueous solutions of SnF₂ and NaF.

Table 2.

Results (study 1) – solutions

NaF solutions
Parts per million (ppm) F in solution	0	12.5	25	50	100	200	400
Volume of titrant (ml)	14.39	10.70	10.03	8.88	8.25	7.80	7.49
% Reduction in dissolution	–	25.7	30.3	38.3	42.7	45.8	47.9

SnF2 solutions
Parts per million (ppm) in solution	Sn	0	38	76	151	227	379	757
	F	0	12	24	49	73	121	243
Volume of titrant (ml)		13.43	9.78	8.33	7.40	5.90	5.13	3.45
% Reduction in dissolution		–	27.2	38.0	44.9	56.1	61.8	74.3

Table 3.

pH stat data (study 2) – dentifrice

Time (minutes)*	Water†	NaF†	Stabilised SnF2†
0.0	0.00	0.00	0.00
1.0	1.43	0.94	0.14
2.0	2.99	1.25	0.14
3.0	3.60	1.41	0.16
4.0	4.04	1.50	0.18
5.0	4.18	1.55	0.18
10.0	4.54	1.70	0.22
15.0	4.70	1.79	0.26
20.0	4.80	1.83	0.30
25.0	4.83	1.88	0.33
30.0	4.89	1.89	0.35

^*Approximate time each measurement was taken.

^†Each data-point represents an average of three independent runs.

Table 4.

Results (Study 2) – dentifrice

Test product	Average volume of titrant (ml)	% Reduction in dissolution	t-Test*
Water	4.89 ± 0.24	–	A
Crest Cavity Protection (NaF)	1.89 ± 0.13	61.3	B
Crest Pro-Health (stabilised SnF2)	0.35 ± 0.04	92.8	C

^*Each mean value grouped according to statistical similarity in Student’s paired t-test.

Figure 3.

Per cent inhibition of hydroxyapatite (HAP) dissolution; SnF₂versus NaF dentifrice.

DISCUSSION

When SnF₂ dentifrices were originally developed, there was little knowledge regarding the relative benefits of stannous and fluoride ions on anticaries efficacy. Study-to-study differences made it difficult to arrive at a consensus as to the mechanism of action for this unique, metal based ingredient. While one study would detect significant differences in the ability of SnF₂ to reduce enamel solubility¹⁶, another would not¹². Researchers recognised that the mere presence of the compound in a dentifrice did not necessarily mean the product would be effective at protecting the enamel against caries acid attack. Some studies comparing fluoride solutions with and without stannous ions noted no differences in performance, suggesting the acid protection benefit was driven predominantly by the fluoride rather than the stannous ion. Studies by Manly¹⁶ demonstrated that in the SnF₂ dentifrices he tested, less than 10% of the stannous ions were being used to provide enamel protection. Wachtel¹² suggested that this level of stannous ion would be insufficient to provide significant protection to tooth surfaces against a prolonged acid challenge in vitro. These results strongly suggest one reason for the inconsistencies in performance of the original SnF₂-containing products was a general lack of understanding and appreciation of the differences in total versus available active in these formulations. It is one thing to put SnF₂ into a dentifrice but it can be quite another thing to keep the SnF₂ stable within the dentifrice, allow it to release from the dentifrice during use and react with tooth surfaces with high levels of bioavailability.

Stabilisation of the SnF₂ has enabled both the long-term viability of dentifrice formulations and the delivery of high levels of bioavailable SnF₂. Compared with the original studies of SnF₂, where less than 10% of the active was believed to be available, the current studies demonstrate that the marketed SnF₂ dentifrice provides protection at least as good as that of a freshly prepared SnF₂ solution. The SnF₂ dentifrice used in Study 2 was a stabilised SnF₂ formulation. Stabilisation of the formula provides significant levels of available active over the projected lifetime of the product – a marked improvement over the original SnF₂ dentifrice available in the 1950s.

Cooley¹¹ proposed that the fluoride ion penetrates the enamel surface, while the tin is deposited as a uniform coating on the tooth surface. Jordan²⁵ demonstrated that SnF₂ can react with tooth mineral to form a stannous fluorophosphate complex that coats the treated surface. The results of the current studies are in agreement with these early perspectives, clearly demonstrating the deposition of an acid resistant barrier layer onto the HAP. The enhanced protection provided by SnF₂ suggests that the outer facing surface is likely composed primarily of stannous, as suggested by Jordan²⁵, with the fluoride component penetrating, or at least positioned toward, the HAP surface¹¹. When NaF is used to treat HAP powder, the reaction product favours formation of fluoridated apatite, with the F⁻ component attaching to the HAP mineral and the Na⁺ dissolving into solution. With SnF₂ treatment, the F again reacts with the HAP, while the Sn portion remains connected to the complex as a protective, barrier shield against acid attack.

Hydroxyapatite powders are particularly useful for modelling dissolution patterns of hard tissue surfaces in the presence of compounds that attach to and protect mineral surfaces against external challenges. In the current studies, powdered apatite was used to demonstrate the ability of SnF₂ and NaF to protect the treated HAP against erosive acid dissolution. This type of model is particularly well-suited for understanding mechanisms of action related to dental erosion processes because dental erosion is a condition characterised by direct dissolution of tooth mineral in an acidic environment. Under the conditions of this model, SnF₂ provided significantly greater protection against erosive acid attack compared with NaF. This particular set of studies compared only the ability of these two agents to attach to mineral surfaces and protect against direct acid challenges over a 30-minute period. Long-term duration of the acid resistance benefit would presumably depend on the tenacity with which the protective agent adheres to mineral surfaces, along with how completely the surface is coated. A separate study focused on both the deposition and retention of stannous ions onto intact, pellicle-coated human enamel surfaces²⁶, as deposition and retention on pellicle-coated surfaces is important to understanding whether the protective benefits remain under more realistic conditions.

The powdered apatite studies reported here demonstrate the ability of SnF₂ to attach to and protect exposed mineral surfaces against acid attack. While it is likely the primary anticaries benefit from SnF₂ comes from its ability to fluoridate subsurface tooth mineral, the surface-facing stannous component provides enhanced resistance against dietary, erosive acids that can lead to dental erosion. Although early studies suggested the ability of SnF₂ to coat and protect tooth mineral against acid challenge, the inconsistencies in results were most likely caused by the instability of formulations available at the time.

CONCLUSION

Results of the current studies suggest a primary mechanism of action for F is via enhancement of acid resistance of tooth mineral, and that resistance is significantly greater after treatment with SnF₂ compared with treatment of tooth mineral with NaF.

Acknowledgements

These studies were funded by The Procter & Gamble Company, Mason, Ohio 45040, USA.

Conflicts of interest

A. A. Baig, S. L. Eversole and M. Lawless are full-time employees at The Procter & Gamble Company, Mason, OH, USA. J. Yan and N. Ji are full-time employees at The Procter & Gamble Company, Beijing, China. R. V. Faller is a retired Principal Scientist from The Procter & Gamble Company, Mason, OH, USA and is now an Associate Professor at the Kornberg School of Dentistry, Temple University, Philadelphia, PA, USA.